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STRUCTURAL PROPERTIES OF ANTIMICROBIAL PEPTIDES ACTING ON BACTERIAL MEMBRANES Boštjan Japelj Lek Pharmaceuticals, Drug Discovery, Ljubljana, Slovenia.

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Presentation on theme: "STRUCTURAL PROPERTIES OF ANTIMICROBIAL PEPTIDES ACTING ON BACTERIAL MEMBRANES Boštjan Japelj Lek Pharmaceuticals, Drug Discovery, Ljubljana, Slovenia."— Presentation transcript:

1 STRUCTURAL PROPERTIES OF ANTIMICROBIAL PEPTIDES ACTING ON BACTERIAL MEMBRANES Boštjan Japelj Lek Pharmaceuticals, Drug Discovery, Ljubljana, Slovenia

2 Antibiotics – miracle drugs Bacterial resistance is becoming a major problem in modern medicine

3 Cationic antimicrobial peptides: - up to 50 aminoacids long - a net positive charge of at least +2 (Arg, Lys) - antimicrobial activity against G-, G+ bacteria, fungi, protozoa, viruses, anticancer activity, effectors of innate immune response - 4 structural classes: ref:

4 ref.: Matsuzaki, K.,. Biochim Biophys Acta, (1-2): p act on membranes and intracellular targets, -advantage: fast acting, resistance is unlikely to develop, able to neutralize bacterial endotoxins and prevent development of sepsis

5 LACTOFERRIN LF11 FQWQRNIRKVR - NH2 C12LF11 lauryl- FQWQRNIRKVR - NH2 P3-55 octanoyl -FWRIRIRR – NH2 Membrane models: -LPS (lipopolysaccharide) : model for baterial membrane -SDS (sodium dodecyl sulphate): model for bacterial membrane -DPC (dodecyl phosphocholine): model for eucaryotic membrane

6 NMR study of LF11 + S-LPS, LF11 + SDS, LF11 + DPC TRNOE between aromatic and aliphatic side chains in 2 mM LF11 upon addition of 1/20 of molar ratio of LPS (b) and LTA (c). The reference NOESY spectrum of LF11 is shown in (a). Spectra were recorded at a mixing time of 150 ms. LF11 + S-LPS: trNOE + k off LF11LPS LF11-LPS CPMG-T2 experiment:

7 Family of 3D structures of LF11 in complex with LPS. ( basic, hydrophobic, LF11 + S-LPS Complex between LF11 and LPS 441 Å 2 of surface area buried polar residues)

8 Comparison of LPS interaction motifs in FhuA (left) 1, LF11 (center) 2 and polymyxin B (right) 3,4 in the same orientation with respect to LPS, which would be in front of the plane of the page Binding motif: LF11 :Phe 1, Arg 5, Lys 9, Arg 11 FhuA :Phe 355, Lys 439, Arg 384, Lys 351 PmxB :Phe 6, Dab 8,9, Dab 3, Dab 1 1 Ferguson, A. D., Hofmann, E., Coulton, J. W., Diederichs, K., and Welte, W. (1998) Science 282: 2215– Japelj, B., Pristovšek, P., Majerle, A., Jerala, R. J Biol Chem, (17): p Pristovšek, P. and J. Kidrič, J Med Chem, (22): Pristovšek, P., Simčič, S., Wraber, B., Urleb, U., J Med Chem, :

9 Comparison of structures LF11+S-LPS, LF11+SDS, LF11+DPC LF11+SDS LF11+S-LPSLF11+DPC

10 N-terminal part of LF11 is protected from fluoresscence quenching Fluorescence quenching LF11 FQWQRNIRKVR-NH F 0, F…Fluorescence emission intensity in the absence and presence of the quencher(Q) [Q]… concentration of the quencher K SV … Stern-Volmer quenching constant

11 C12LF11: -acylation enhances antimicrobial activity against G- and G+ bacteria CD spectra of LF11 and C12LF11 in DPC micelles Family of structures of C12LF11 in DPC 1 -acylation stabilizes secondary structure 1 Japelj, B., Zorko, M., Majerle, A., Pristovšek, P.,et al.. JACS, (2007), 129:

12 OCTANOYL-F W R I R I R R – NH P3-55:

13 Circular dichroism spectra

14 Backbone conformation of P3-55 in DPC Structure of P3-55 in DPCStructure of P3-55 in SDS

15 Positioning and orientation of P3-55 in micelles (NMR experiments using paramagnetic probes 5-DSA and 16-DSA) doxyl group

16 reference5 - DSA NOESY TOCSY

17 Normalized I/I ref ratios of H N -H cross peaks in NOESY spectrum P3-55 in SDS P3-55 in DPC

18 Order parameter tensor elements of DPC micelle for the simulation of DPC micelle a) and DPC micelle + P3-55 b). –S CD =2/3S xx + 1/2S yy DPC DPC + P3-55 Molecular dynamics of P3-55 in DPC Total energy (left) and temperature (right) of the system (P DPC SOL + 4 Cl-) during first 2 ns of simulation time [ps] Ratios between princpal moments of inertia of DPC during simulation of P3-55 in DPC. Principal moments of inertia are shown in the table. *moments of inertia in units 10 4 amu nm 2. Asymetry parameter,, defined as = (2I 1 -I 2 -I 3 )/(I 1 +I 2 +I 3 ) * Radial density of P3-55 in complex with DPC micelle. DPC coordinates were taken fromTieleman, D.P., et al. J. Phys Chem. B. 2000, 104:

19 Outer membrane Cytoplasmic membrane Mechanism of interaction of ANEPID peptides with the membrane of Gram-negative bacteria.

20 Acknowledgements: Andreja Majerle, Primož Pristovšek, Mateja Zorko, Roman Jerala (NIC, Ljubljana) Miha Kotnik, Katja Kristan, Drago Kuzman, Andrej Preželj, Jan Humljan, Petra Igličar, Vjekoslava Car, Uroš Urleb (Lek, Drug Discovery, Ljubljana) co-workers from the EU project ANEPID (Antimicrobial Endotoxin-neutralazing Peptides to Combat Infectious Deseases): Dagmar Zweytick, Karl Lohner (Graz) Guillermo Martinez de Tejada, Ignacio Moriyon, Susana Sanchez-Gomez (Pamplona) Sylvie E. Blondelle (San Diego, CA) Klaus Brandenburg, Jörg Andrä (Borstel)


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